Disease-resistant plants and method of constructing the same

ABSTRACT

It is the object of the present invention to provide disease-resistant plants which have been transformed to cause an effective defense reaction, and methods for producing the same.  
     The present invention provides expression cassettes comprising a promoter capable of promoting a constitutive, inducible, or organ- or phase-specific gene expression, and a gene, under the control of said promoter, encoding an elicitor protein.

FIELD OF THE INVENTION

[0001] The present invention relates to methods for producingdisease-resistant plants, gene expression cassettes for producingdisease-resistant plants, and transgenic, disease-resistant plantsproduced by the method.

BACKGROUND OF THE INVENTION

[0002] Plant defense against pathogens differs in its mechanism fromthat observed in animals. For example, there is known in higher plants ahypersensitive response (HR) mechanism which involves a dynamicresistance reaction to pathogen invasion. When a pathogen invades aplant, plant cells at a site of invasion die in response, wherebypathogens are trapped locally. This reaction is known to be induced as aresult of either an incompatible host-pathogen interaction or anon-host-pathogen interaction. Such cell suicide can be understood interms of a localized, programmed cell death (Dangl et al.: Plant Cell 8:1973-1807 (1996)). In addition to the mechanism involving HR, otherdefense reactions, including generation of active oxygen species,reinforcement of a cell wall, production of phytoalexin and biosynthesisof defense-related proteins such as PR proteins, are also known(Hammond-Kosack and Jones: Plant Cell 8: 1773-1791 (1996)). Further, inaddition to such localized defense responses, there is known to takeplace in many cases a defense reaction spreads whereby PR proteinsaccumulate also in non-infected parts of a plant, whereby resistance isimparted to the entire plant. This mechanism is referred to as systemicacquired resistance (SAR) and continues for several weeks or longer. Asa result, the entire plant is made resistant to secondary infection(Sticher et al.: Annu. Rev. Phytopathol. 35: 235-270 (1997)).

[0003] A first reaction of a plant of switching on a highly organizeddefense reaction such as outlined above is the recognition by the plantof a molecule called an “elicitor” directly or indirectly produced by aninvading pathogen. Additionally, complex signal cascades including thesubsequent rapid generation of active oxygen species and reversibleprotein phosphorylation are considered to be important as initialreactions of the defense response (Yang et al.: Genes Dev. 11: 1621-1639(1997)). There are a wide variety of elicitors, including so-callednonspecific elicitors e.g. oligosaccharides which are products bydegradation of cell wall components of many fungi includingchitin/chitosan and glucan, or oligogalacturonic acids derived from aplant cell wall, variety-specific elicitors e.g. avirulence geneproducts of pathogens such as AVR 9 (Avr gene products), and elicitorswith an intermediate specificity such as elicitin (Boller: Annu. Rev.Plant Physiol. Plant Mol. Biol. 46: 189-214 (1995)).

[0004] Harpin is a bacterium-derived protein elicitor which induceshypersensitive cell death in a non-host plant (Wei et al.: Science 257:85-88 (1992), He et al.: Cell 73: 1255-1266 (1993)). Harpin(harpin_(Ea)) has been purified as a first bacterium-derived HR-inducingprotein from Erwinia amylovora Ea321, a pathogen of pear and apple, andEscherichia coli transformed with a cosmid containing the hrp genecluster, and an hrpN gene encoding Harpin has been cloned (Wei et al.:Science 257: 85-88 (1992)). Thereafter, harping_(pss) encoded by hrpZgene has been identified and characterized from Pseudomonas syringae pv.syringae 61, a pathogen of a bean, by screening an Escherichia coliexpression library with an activity of inducing HR to a tobacco leaf asan index (He et al.: Cell 73: 1255-1266 (1993), and Japanese PatentApplication Domestic Announcement No. 1996-510127). The homology betweenthese two harpins is low, and a relatively high homology is found onlyin 22 amino acids. Moreover, the role of a harpin in pathogenicity hasnot been made clear. In addition to these, as a third protein, PopAprotein (which PopA encodes) is identified from Pseudomonas solanacearumGMI1000, a pathogen of a tomato, as a protein inducing HR to a non-hosttobacco (Arlat et al.: EMBO. J. 13: 543-553 (1994)). Though PopA gene islocated on the outside of hrp cluster, differing from hrpN and hrpZ,they are identical in that they are under the control of an hrp regulon.The above three proteins are glycine-rich, heat stable proteins, induceHR to a non-host tobacco and are secreted extracellularly at least invitro in a manner of depending upon hrp protein. In addition to theseare reported HrpW protein from Pseudomonas syringae pv. tomato DC3000 asa protein having the same function (Charkowski et al.: J. Bacteriol.180: 5211-5217 (1998)), hrpZ_(pst) and hrpZ_(psg) proteins asharpin_(pss) homologues (Preston et al.: Mol. Plant-Microbe. Interact.8: 717-732 (1995)), and harpin_(Ech) (Bauer et al.: Mol. Plant-Microbe.Interact. 8: 484-491 (1995)) and hrpN_(Ecc) protein (Cui et al.: Mol.Plant-Microbe. Interact. 9: 565-573 (1996)) as harpin_(Ea) homologues.

[0005] It has been made apparent from studies upon various metabolicinhibitors that the formation of localized necrosis spots with harpin isnot so-called necrosis due to the cytotoxicity of harpin but a celldeath resulting from a positive response on the plant side (He et al.:Mol. Plant-Microbe. Interact. 7: 289-292 (1994), and He et al.: Cell 73:1255-1266 (1993)), and this hypersensitive cell death is thought to be atype of programmed cell death (Desikan et al.: Biochem. J. 330: 115-120(1998)). The addition of harpin_(pss) into a cell culture of Arabidopsisinduces a homologue of gp91-phox, a constituent of NADPH oxidase, whichis thought to have an important role in the oxidative burst as aninitial reaction of a disease-resistant reaction, (J. Exp. Bot. 49:1767-1771 (1998)), and mitogen-activated protein (MAP) kinase (Desikanet al.: Planta. 210: 97-103 (1999)). Moreover, a harpin can impartsystemic acquired resistance (SAR) to a plant. For example, SARmeditated by salicylic acid and an NIM gene can be induced to anArabidopsis plant by artificially injecting harpin_(Ea) into the plantcells (Dong et al.: The Plant J. 20: 207-215 (1999)), and Harpin_(pss)can induce SAR to a cucumber and impart a wide spectrum of resistance tofungi, viruses and bacteria (Strobel et al.: Plant J. 9: 431-439(1996)).

[0006] Thus, there are reports about artificially injecting or sprayingpurified harpin into a plant and analyzing the induction of ahypersensitive cell death and an acquired resistance reaction (JapanesePatent Application Domestic Announcement No. 1999-506938, Strobel etal.: Plant J. 9: 431-439 (1996), and Dong et al.: The Plant J. 20:207-215 (1999)). However, there is no report about introducing a geneencoding an elicitor protein such as a harpin into a plant to produce atransgenic plant and analyzing it.

SUMMARY OF THE INVENTION

[0007] It has been anticipated that, when a gene encoding an elicitorprotein such as harpin is introduced into a plant, the plant willexpress an elicitor protein at a certain amount, even in a normal statewith no pathogen, or that it will also express an elicitor protein in acertain amount in organs other than those invaded with a disease, and asa result, various unintended reactions occur to prevent the plant fromgrowing normally. The object of the present invention is therefore toprovide a disease-resistant transgenic plant which has been transformedto induce a proper defense reaction, and to provide a method forproducing the same.

[0008] The present inventors have engaged in studies assiduously, and asa result have found that a transgenic tobacco with hrpZ gene ofPsedomonas syringae pv. syringae LOB2-1 introduced thereinto induceshypersensitive-response-like localized necrosis spots in response to theinoculation of a powdery mildew fungi (Erysiphe cichoracearum) to becomeresistant, which has led to the completion of the present invention.Surprisingly, a plant grew normally when cell-death-inducing harpin wasexpressed with a constitutive promoter (cauliflower mosaic virus 35S RNAgene promoter) capable of promoting expression in cells of the wholebody. In addition, a hypersensitive cell-death-like reaction was inducedonly after inoculation with a pathogen. Further, the present inventorshave found that a transgenic rice with the same hrpZ gene introducedthereinto becomes blast (Magnaporthe grisea)-resistant, thus showing thegeneral-applicability of the present invention.

[0009] The present invention provides a transgenic, disease-resistantplant which has been transformed with an expression cassette comprisinga promoter capable of promoting a constitutive, inducible, or organ- orphase-specific gene expression and a gene encoding an elicitor proteinunder the control of said promoter, wherein said plant is capable ofeffecting the constitutive, inducible, or organ- or phase-specificexpression of the elicitor protein in an amount effective for inducing adefense reaction.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010]FIG. 1 shows the constructs constructed and introduced into plantsin the present invention.

[0011]FIG. 2 is a photograph showing exemplary of the detection resultsusing Western analysis for harpin_(pss) accumulation in transgenictobacco and rice of the T₀ generation. PC represents harpin_(pss)expression in Escherichia coli as a control.

[0012]FIG. 3 is a photograph showing the appearances of localizednecrosis spots occurring in a transgenic tobacco of the T₁ generation.A: PALL-hrpZ-introduced individual (5th day after inoculation, harpinexpression level: ++), B: 35S-hrpZ-introduced individual (7th day afterinoculation, harpin expression level: ++)

[0013]FIG. 4 is a photograph showing the resistance of a transgenictobacco of the T₁ generation against powdery mildew. (Right:35S-hrpZ-introduced individual, harpin expression level: ++, Left: SR1as a control, 11th day after inoculation in both)

DETAILED DESCRIPTION OF THE INVENTION

[0014] The present invention also provides methods for producingtransgenic, disease-resistant plants capable of effecting theconstitutive, inducible, or organ- or phase-specific expression of anelicitor protein in an amount effective for inducing a defense reaction.Such methods comprise the steps of: (a) obtaining transgenic plant cellswith expression cassettes comprising a promoter capable of promoting aconstitutive, inducible, or organ- or phase-specific gene expression anda gene encoding an elicitor protein under the control of said promoter;and (b) regenerating a complete plant from said transgenic plant cell.

[0015] The present invention also provides expression cassettes capableof being employed for producing a transgenic, disease-resistant plants.Such expression cassettes comprise at least: (a) a promoter capable ofpromoting a constitutive, inducible, or organ- or phase-specific geneexpression; and (b) a gene, under the control of said promoter, encodingan elicitor protein. “Elicitor” is a general term used for substancesinducing defense reactions in plants, and including heavy metal ions,and cell wall components of pathogens or plants, in addition toproteins. The term “elicitor” as used in the present specificationrefers to a protein elicitor unless otherwise specified.

[0016] The term “elicitor protein” as used in the present invention canbe any protein capable of inducing a proper defense reaction in a plantto be transformed, and preferably a protein possessing ahypersensitive-response-inducing activity against pathogenicmicroorganisms. It includes harpin and a harpin-like protein having thesame function as harpin. “Harpin” is a protein expected to be introducedinto a plant in a manner of depending upon hrp gene though the Type IIIsecretion mechanism, and includes, in addition to harpin_(pss), (He etal.: Cell 73: 1255-1266 (1993), and Japanese Patent Application DomesticAnnouncement[kohyo] No. 510127/96), harpin_(Ea) (Wei et al.: Science257: 85-88 (1992), and Japanese Patent Application DomesticAnnouncement[kohyo]No. 506938/99), PopA (Arlat et al.: EMBO. J. 13:543-553 (1994)), and hrpW protein (Charkowski et al.: J. Bacteriol. 180:5211-5217 (1998). Additionally the protein possessing ahypersensitive-response-inducing activity can be, for example, (a) aprotein consisting of the amino acid sequence of SEQ. ID No. 2; (b) aprotein consisting of an amino acid sequence derived from the amino acidsequence of SEQ. ID No. 2 by deletion, substitution, addition orinsertion of one or more amino acids, and possessing ahypersensitive-response-inducing activity; or (c) a protein consistingof an amino acid sequence being at least 50% (preferably at least 80%,more preferably at least 90%, and still more preferably at least 97%)homologous to the amino acid sequence of SEQ. ID No. 2, and possessing ahypersensitive-response-inducing activity. A protein consisting of theamino acid of SEQ ID No. 2 is novel. Hence, the present inventionprovides one of the following proteins: (a) a protein consisting of theamino acid sequence of SEQ. ID No. 2; (b) a protein consisting of anamino acid sequence derived from the amino acid sequence of SEQ. ID No.2 by deletion, substitution, addition or insertion of one or more aminoacids, and possessing a hypersensitive-response-inducing activity; and(c) a protein consisting of an amino acid sequence being at least 97%homologous to the amino acid sequence of SEQ. ID No. 2, and possessing ahypersensitive-response-inducing activity (but known proteins themselvesare excluded from the scope of the present invention).

[0017] By “Homology” referred to in connection with amino acid sequencesin the present specification is meant a degree of identification ofamino acid residues constituting each sequence between sequences to becompared. In homology, the existence of a gap(s) and the nature of anamino acid(s) are taken into consideration (Wilbur, Proc. Natl. Acad.Sci. USA 80: 726-730 (1983) and the like). To calculate homology,commercially available software such as BLAST (Altschul: J. Mol. Biol.215: 403-410 (1990), and FASTA (Peasron: Methods in Enzymology 183:63-69 (1990)) can be employed.

[0018] The description “deletion, substitution, addition or insertion ofone or more amino acids” as used in the present specification inconnection with an amino acid sequence in the means that a certainnumber of an amino acid(s) are substituted etc. by any well knowntechnical method such as site-specific mutagenesis, or naturally. Thenumber is, for example, up to ten, and is preferably from 3 to up to 5.

[0019] A gene encoding an elicitor protein to be employed in theexpression cassette of the present invention can easily be isolated bymethods well-known to those skilled in the art.

[0020] The gene encoding an elicitor protein can be, for example, (a) aDNA molecule consisting of the nucleotide sequence of SEQ. ID No. 1; (b)a DNA molecule consisting of a nucleotide sequence derived from thenucleotide sequence of SEQ. ID No. 1 by deletion, substitution, additionor insertion of one or more nucleotides, and encoding a proteinpossessing a hypersensitive-response-inducing activity; (c) a DNAmolecule consisting of a nucleotide sequence being hybridizable with aDNA molecule consisting of the nucleotide sequence complementary to thenucleotide sequence of SEQ. ID No. 1 under stringent conditions, andencoding a protein possessing a hypersensitive-response-inducingactivity; or (d) a DNA molecule consisting of a nucleotide sequencebeing at least 50% (preferably at least 80%, more preferably at least90%, and still more preferably at least 97%) homologous to thenucleotide sequence of SEQ. ID No. 1, and encoding a protein possessinga hypersensitive-response-inducing activity. A DNA molecule consistingof the nucleotide sequence of SEQ ID No. 1 is novel. Hence, the presentinvention also provides a gene consisting of one of the following DNAmolecules: (a) a DNA molecule consisting of the nucleotide sequence ofSEQ. ID No. 1; (b) a DNA molecule consisting of a nucleotide sequencederived from the nucleotide sequence of SEQ. ID No. 1 by deletion,substitution, addition or insertion of one or more nucleotides, andencoding a protein possessing a hypersensitive-response-inducingactivity; (c) a DNA molecule consisting of a nucleotide sequence beinghybridizable with a DNA molecule consisting of the complementarynucleotide sequence to the nucleotide sequence of SEQ. ID No. 1 understringent conditions, and encoding a protein possessing ahypersensitive-response-inducing activity; or (d) a DNA moleculeconsisting of a nucleotide sequence being at least 50% homologous to thenucleotide sequence of SEQ. ID No. 1, and encoding a protein possessinga hypersensitive-response-inducing activity (but known genes themselvessuch as hrpZ gene of Pseudomonas syringae pv. syringae 61 are excludedfrom the scope of the present invention). To calculate homology inconnection with nucleotide sequences, commercially available softwarecan be employed.

[0021] By “deletion, substitution, addition or insertion of one or morenucleotides” in connection with a nucleotide sequence in the presentspecification is meant that a certain number of a nucleotide(s) aresubstituted etc. by a well-known technical method such as asite-specific mutagenesis or naturally. The number is, for example, upto ten, preferably from 3 to up to 5. By “stringent conditions” referredto in the present specification is meant hybridization conditionswherein the temperature is at about 40° C. or above and that the saltconcentration is of about 6×SSC (1×SSC=15 mM sodium citrate buffer; pH:7.0; 0.15 M sodium chloride; 0.1% SDS), preferably at about 50° C. orabove, more preferably at about 65° C. or above.

[0022] The promoter to be employed in the present invention can be anypromoter capable of functioning as a promoter for a gene encoding anelicitor protein in a plant to be transformed. In the present invention,a promoter capable of promoting a constitutive, inducible, or organ- orphase-specific gene expression can be employed.

[0023] By “promoter promoting a constitutive gene expression (oftenreferred to as a “constitutive promoter”)” is meant a promoter whoseorgan specificity and/or phase specificity are (is) not high inconnection with the transcription of the gene. Examples of theconstitutive promoter include cauliflower mosaic virus 35S promoter,ubiquitin promoter (Cornejo et al.: Plant Mol. Biol. 23: 567-581(1993)), actin promoter (McElroy et al.: Plant Cell 2: 163-171 (1990)),alpha tubulin promoter (Carpenter et al.: Plant Mol. Biol. 21: 937-942(1993)) and Sc promoter (Schenk et al.: Plant Mol. Biol. 39: 1221-1230(1999)). In a transgenic plant, the expression cassette promoting theconstitutive expression of an elicitor protein includes, for example, aknown promoter that is known as a constitutive promoter.

[0024] By “promoter promoting an inducible gene expression (oftenreferred to as an “inducible promoter”)” is meant a promoter whichinduces transcription by physical or chemical stimulation, such aslight, disease, injury or contact with an elicitor. Examples of theinducible promoter include pea PAL promoter, Prp1 promoter (JapanesePatent Application No. 1998-500312), hsr203J promoter (Pontier et al.:Plant J. 5: 507-521 (1994)), EAS4 promoter (Yin et al.: Plant Physiol.115: 437-451 (1997)), PR1b1 promoter (Tornero et al.: Mol. PlantMicrobe. Interact. 10: 624-634 (1997)), tap1 promoter (Mohan et al.:Plant Mol. Biol. 22: 475-490 (1993)) and AoPR1 promoter (Warner et al.:Plant J. 3: 191-201 (1993)). In a transgenic plant, the expressioncassette promoting an inducible elicitor protein expression includes,for example, a known promoter known as an inducible promoter.

[0025] By “promoter promoting an organ-specific gene expression (oftenreferred to as an “organ-specific promoter”)” is meant a promotergiving, to the transcription of the gene, a specificity to an organ,such as a leaf, a root, a stem, a flower, a stamen and a pistil.Examples of the organ-specific promoter include a promoter promoting ahigh gene expression in green tissues of a photosynthesis-related gene,such as PPDK (Matsuoka et al.: Proc. Natl. Acad. Sci. USA 90: 9586-9590(1993)), PEPC (Yanagisawa and Izui: J. Biochem. 106: 982-987 (1989) andMatsuoka et al.: Plant J. 6: 311-319 (1994)) and Rubisco (Matsuoka etal.: Plant J. 6: 311-319 (1994)). In a transgenic plant, the expressioncassette promoting an organ-specific elicitor protein expressionincludes, for example, a known promoter that is known as anorgan-specific promoter.

[0026] By “promoter promoting a phase-specific gene expression (oftenreferred to as a “phase-specific promoter”)” is meant a promoter giving,to the transcription of the gene, a phase specificity to a phase, suchas a initial, middle and later growth phase. Examples of thephase-specific promoter include a promoter functioning specifically inaged leaves such as SAG12 promoter (Gan and Amashino: Science 270:1986-1988 (1985)).

[0027] Vectors for sub-cloning each DNA fragment as a component of theexpression cassette of the present invention can be simply prepared byconnecting an intended gene into a vector for recombination (plasmidDNA) available in the art by any common technique. Specific examples ofsuitable vectors include plasmids derived from Escherichia coli, such aspBluescript, pUC18, pUC19 and pBR322, but are not limited only to theseplasmids.

[0028] As a vector for introducing the expression cassette of thepresent invention into a plant to be transformed, a vector fortransforming plants can be used. The vectors for plants are notparticularly limited, so far as they are capable of expressing theconcerned gene and producing the concerned protein in a plant cell, andexamples thereof include pBI221, pBI121 (both being manufactured byClontech) and vectors derived therefrom. In addition, for thetransformation of a monocotyledonous plant in particular, there can beexemplified pIG121Hm, pTOK233 (both by Hiei et al.: Plant J. 6: 271-282(1994)), pSB424 (Komari et al.: Plant J. 10: 165-174 (1996)),superbinary vector pSB21 and vectors derived therefrom. A recombinationvector having the expression cassette of the present invention can beconstructed by introducing a gene encoding an elicitor protein into anyof these known vectors (if required, a promoter region being recombined)by a procedure known well to those skilled in the art. For example, arecombinant vector having an expression cassette comprising aconstitutive promoter and hrpZ gene can be constructed by integratinghrpZ gene into superbinary vector pSB21. A recombinant vector having anexpression cassette comprising an inducible promoter and hrpZ gene canbe constructed by removing the existing promoter from the aboverecombinant vector and integrating an inducible promoter in place.

[0029] A plant-transforming vector preferably comprises at least apromoter, a translation initiator codon, a desired gene (a DNA sequenceof the invention of the present application or a part thereof), atranslation termination codon and a terminator. Moreover, it maycomprise a DNA molecule encoding a signal peptide, an enhancer sequence,a non-translation region on the 5′ side and the 3′ side of the desiredgene and a selection marker region as appropriate. Examples of markergenes include antibiotic-resistant genes such as tetracyclin,ampicillin, kanamycin or neomycin, hygromycin or spectinomycin; andgenes such as luciferase, β-galactosidase, β-glucuronidase(GUS), greenfluorescence protein (GFP), β-lactamase and chloramphenicol acetyltransferase (CAT).

[0030] As methods for introducing a gene into a plant can be mentioned amethod employing an agrobacterium (Horsch et al.: Science 227: 129(1985), Hiei et al.: Plant J. 6: 271282 (1994)), a leaf disc method(Horsch et al.: Science 227: 1229-1231 (1985), an electroporation method(Fromm et al.: Nature 319: 791 (1986)), a PEG method (Paszkowski et al.:EMBO. J. 3: 2717 (1984)), a micro-injection method (Crossway et al.:Mol. Gen. Genet. 202: 179 (1986)) and a minute substance collisionmethod (McCabe et al.: Bio/Technology 6: 923 (1988)), but any method forintroducing a gene into a desired plant may be employed without anyparticular limitation. Of these methods for transfection, a methodcomprising transferring a vector into an agrobacterium by mating andthen infecting a plant with the agrobacterium is preferred. Methods forinfection is also well-known to those skilled in the art. Examplesinclude a method comprising damaging a plant tissue and infecting itwith a bacterium; a method comprising infecting an embryo tissue(including an immature embryo) of a plant with the bacterium; a methodcomprising infecting with a callus; a method comprising co-culturingprotoplasts and the bacterium; and a method comprising culturing afragment of a leaf tissue together with the bacterium (leaf discmethod).

[0031] Successfully transformed cells can be selected from other cellsby employing an appropriate marker as an index or examining theexpression of a desired trait. The transformed cell can further bedifferentiated employing a conventional technique to obtain a desiredtransgenic plant.

[0032] Analysis of the resultant transformant can be performed byemploying various methods that are well-known to those skilled in theart. For example, oligonucleotide primers can be synthesized accordingto the DNA sequence of the introduced gene, and the chromosome DNA ofthe transgenic plant can be analyzed by PCR employing the primers. Inaddition, the analysis can be performed on the basis of the existence ofmRNA corresponding to the introduced gene and the existence of theprotein expression. Moreover, the analysis can be performed on the basisof the appearance of the plant (for example, in the case oftransformation with a gene encoding a protein capable of inducinglocalized necrosis spots, the presence of localized necrosis spots, orthe size, number and the like of the localized necrosis spots), diseaseresistance (for example, the existence of resistance or its degree uponcontacting the plant with a pathogen) and the like.

[0033] In the transgenic plant of the present invention, a constitutive,inducible, or organ- or phase-specific expression of an elicitor proteinin an amount effective for inducing a defense reaction can be achieved.The amount effective for inducing a defense reaction is such an amountthat the expressed elicitor protein can induce at least a localizeddefense-related reaction (for example, induction of a hypersensitivecell death (localized necrosis)) to the plant. Preferably, the amount issuch that the defense reaction extends to the whole body of the plant,and as a result, the whole plant becomes resistant (systemic acquireddisease-resistant). Moreover, preferably, the amount is not so largethat causes death of the localized tissue having the necrosis spots as aresult of the localized necrosis spots becoming too large.

[0034] Moreover, in the transgenic plant of the present invention, anelicitor protein is preferably expressed in an amount which, while beingeffective for inducing a defense reaction in response to stimulationsuch as the invasion of a pathogen, does not, under normal conditions,remarkably prevent the growth of the plant due to the negligible or lowexpression, if any. For example, in the case of employing harpin_(pss)as an elicitor protein, usually no harpin_(pss) is expressed, or isexpressed only in an amount that does not allow localized necrosis spotsto cause the death of the organ, and preferably it is expressed in anamount that induces a hypersensitive response at the time of theinvasion of a pathogen. Further, it is preferably expressed in such anamount that, even if a pathogen invades to cause harpin_(pss) toaccumulate, localized necrosis spots are hardly observable by the nakedeye, but the whole body acquires a systemic disease-resistannce.

[0035] In order to induce such a proper defense reaction, for example, apromoter capable of promoting an inducible gene expression is employed.Hence, in one embodiment of the present invention, an inducible promoterand a harpin gene are combined.

[0036] In addition, a proper defense reaction can be accomplished notonly in the case of employing an inducible promoter but also in the caseof employing a constitutive promoter. Hence, in another embodiment ofthe present invention, a constitutive promoter and a harpin gene areused in combination. In this embodiment, as a mechanism of theoccurrence of a proper defense reaction, it is considered that anelicitor protein, for example, harpin_(pss), is recognized at theoutside of cell membranes or on the cell wall of plant cells, and hence,harpin_(pss) accumulating in cytoplasm is not recognized by plant cellsuntil degradation of cells occurs due to invasion of fungus, and as aresult, the hypersensitive response appears after the inoculation of thepathogen or it is deduced that there exists a further factor which isrelated to the inoculation of a pathogen in the mechanism of theoccurrence of the elicitor activity of harpin_(pss).

[0037] The transgenic plants of the present invention include atransgenic, powdery mildew-resistant tobacco which has been transformedwith an expression cassette comprising a constitutive or induciblepromoter and a gene, under the control of said promoter, encoding anelicitor protein such as harpin_(pss), or a transgenic, blast-resistantrice which has been transformed with an expression cassette comprising aconstitutive promoter and a gene, under the control of the promoter,encoding an elicitor protein such as harpin_(pss).

[0038] It is thought that the present invention can be applied to plantsother than rice and tobacco described in the examples to be describedlater. Examples of such plants include, as crops, wheat, barley, rye,corn, sugar cane, sorghum, cotton, sunflower, peanut, tomato, potato,sweet potato, pea, soybean, azuki bean, lettuce, cabbage, cauliflower,broccoli, turnip, radish, spinach, onion, carrot, eggplant, pumpkin,cucumber, apple, pear, melon, strawberry and burdock; and, as ornamentalplants, arabidopsis thaliana, petunia, chrysanthemum, carnation,saintpaulia and zinnia. The “transgenic plants” referred to in thepresent invention include not only transgenic plants (To generation)obtained by obtaining a transgenic plant cell according to the method ofthe present invention and regenerating, from said plant cell, a completeplant, but also later-generation (T₁ generation and the like) plantsobtained from said transgenic plants so far as the disease-resistanttrait is contained. In addition, the “plants” referred to in the presentinvention include, unless otherwise spcified, in addition to plants(individuals), seeds (including germinated seeds and immature seeds),organs or parts thereof (including a leaf, a root, a stem, a flower, astamen, a pistil and pieces thereof), a plant culture cell, a callus anda protoplast.

[0039] The diseases analyzed in the following examples are tobaccopowdery mildew and rice blast, but as other diseases of tobacco therecan be mentioned wildfire, bacterial wilt and TMV; and as other diseasesof rice there can be mentioned sheath blight disease and bacterial leafblight disease. According to the method for producing adisease-resistant plant of the present invention, it is possible toimpart resistance in plants to these diseases.

EXAMPLES Example 1 Cloning of HrpZ Gene

[0040] A pair of primers for amplifying the open leading frame of hrpZgene were synthesized in reference to the nucleotide sequence of thereported hrpZ gene of Pseudomonas syringae pv. syringae 61 (He et al.:Cell 73: 1255-1266 (1993)), and Japanese Patent Application DomesticAnnouncement[Kohyo] No. 1996-510127): Hrp1: AAA ATC TAG AAT GCA GAG TCTCAG TCT TAA Hrp2: AAA AGT CGA CTC AGG CTG CAG CCT GAT TGC

[0041] Employing these primers, PCR was performed with a DNA molecule ofa cosmid clone containing an hrp cluster derived from Pseudomonassyringae pv. syringae LOB2-1 (a casual agent for bacterial blight oflilac) (Inoue and Takikawa: J. Gen. Plant Pathol. 66: 238-241 (2000)) asa template. PCR was performed under the following conditions: the amountof a reaction solution: 20>1; each primer: 0.5 μM; dNTP: 0.2 mM; 1×ExTaqbuffer; ExTaq DNA polymerase (from Takara Shuzo): 1U; once at 95° C. for5 minutes, then 30 cycles at 94° C. for 30 seconds, at 60° C. for 30seconds and at 72° C. for 2 minutes, and once at 72° C. for 10 minutes.The PCR product was ligated to a vector pCR2.1 (from Invitrogen) usingTakara ligation kit (from Takara Shuzo) and transformed into anEscherichia coli TB1 strain. As a result of determining the entirenucleotide sequence of the PCR product, it consisted of 1029 bp in thelength, longer than the reported hrpZ gene (He et al.: Cell73:1255-1266(1993)) by three bases (one amino acid), and showed ahomogoly of 96.7% in nucleotides and a homology of 96.5% in amino acids.The reason that the nucleotide sequences are not completely the same isthought to be due to a variation among the pathover. The nucleotidesequence of the cloned hrpZ gene is shown in SEQ. ID No. 1 and thededuced amino acid sequence obtained therefrom is shown in SEQ. ID No.2, respectively.

Example 2 Expression in an Escherichia coli and Production of anAntibody

[0042] The above plasmid with an hrpZ gene integrated into pCR2.1 wasdigested with restriction enzymes BamHI and SaII, and was subjected toelectrophoresis on 0.7% agarose to separate a fragment of about 1.1 kb.This fragment was ligated to an expression vector pQE31 (from QIAGEN)digested with the same enzymes and transformed into Eschrichia coli M15strain. The thus obtained Eschrichia coli was cultured in an LB mediumin the presence of 1 mM of IPTG at 37° C., harpin_(pss) was accumulatedas insoluble fraction. Since this protein showed poor adsorption to anickel resin adsorbent, the purification of harpin_(pss) was conductedin the following procedure. The Eschrichia coli M15 strain having thepQE31 vector with the hrpZ gene integrated thereinto was cultured in 2ml of an LB medium containing 100 mg/l of ampicillin and 25 mg/l ofkanamycin at 37° C. overnight, and transferred into 250 ml of the LBmedium and cultured for about three hours; then 1 mM of IPTG was addedthereto and the culture was further conducted at 37° C. for 4 hours.Cells were collected by centrifugation, the insoluble fraction wasdissolved in 4 ml of an eluation buffer (8 M urea, 0.1 M sodiumdihydrogen phosphate, 0.01 M Tris, pH 8.0), and a supernatant liquid wasobtained by centrifugation and subjected to electrophoresis on a 12.5%acrylamide gel containing 0.1% SDS, and then stained with CoomassieBrilliant Blue to cut a band appearing at around 40 kDa. The gel was cutinto small pieces, and an elution buffer (1% SDS, 0.02 M Tris HCl, pH of8.0) was added thereto in an amount ten times the volume of the gel, andshaken for three days. The supernatant was transfered to a dialysismembrane with a cut off molecular weight of 6,000 to 8,000, and thedialysis was conducted with 80% acetone as an external liquid once for 4hours and once overnight. The whole content in the dialysis tube wasmoved into an Eppendorf tube, subjected to centrifugation to discard thesupernatant, and the pellet was dried to obtain a purified harpin_(pss)preparation. 3 mg of the purified harpin_(pss) was sent to SawadyTechnology for the production of an antibody (anti-rabbit harpin_(pss)serum).

Example 3 Construction of a Gene and Transformation of a Plant

[0043] The hrpZ gene integrated into pCR2.1 was excised from the vectorby digestion with restriction enzymes XbaI and SacI (from Takara Shuzo).On the other hand, superbinary vector pSB21 (35S-GUS-NOS, Komari et al.:Plant J. 10: 165174 (1996)) was digested with the same enzymes to removethe GUS gene, and the hrpZ gene was integrated thereinto. According tothe above procedure, a construct named 35S-hrpZ (35S promoter-hrpZgene-NOS terminator) was constructed. The cauliflower mosaic virus 35Spromoter is a promoter capable of constitutively promoting a highexpression, and it is anticipated that rice and tobacco transformed withthis construct will accumulate harpin_(pss), the hrpZ gene product, inthe whole body.

[0044] pSB21 was digested with restriction enzymes HindIII and XbaI toremove the 35S promoter, and a 0.9 kb fragment of corn PPDK promoter(Taniguchi et al.: Plant Cell Physiol. 41: 42-48 (2000)) was integratedthereinto. The resulting plasmid was digested with XbaI and SacI toremove the GUS gene, and then the above-described hrpZ XbaI-SacIfragment was inserted thereinto. Thus, PPDK-hrpZ (PPDK promoter-hrpZgene-NOS terminator) was constructed. The corn PPDK promoter is apromoter capable of promoting a strong expression in photosynthesisorgans such as mesophyl cells (Taniguchi et al.: Plant Cell Physiol. 41:42-48 (2000)), and it is anticipated that rice plants transformed withthis construct will accumulate harpin_(pss), the hrpZ gene product, ingreen organs (leaves).

[0045] PAL promoter was cloned as below. Plasmid DNA was extracted fromagrobacterium LBA4404 strain (gifted from Prof. Shiraishi of OkayamaUniversity) having a construct containing PSPAL1 (PSPAL1 promoter-GUSgene-NOS terminator) (Yamada et al.: Plant Cell Physiol. 35: 917-926(1994), and Kawamata et al.: Plant Cell Physiol. 38: 792-803 (1997)). Onthe other hand, a reverse primer and two forward primers were designedon the basis of the nucleotide sequence of the reported PSPAL1 promoter(Patent: JP 1993153978-A 1 22-Jun.-1993; TAKASAGO INTERNATL. CORP.):PALRVXba: GGG GTC TAG AAT TGA TAC TAA AGT AAC TAA TG PALFFHin: TTG GAAGCT TAG AGA TCA TTA CGA AAT TAA GG PALFSHin: CTA AAA GCT TGG TCA TGC ATGGTT GCT TC

[0046] A promoter region (PAL-S) of about 0.45 kb in the upstream of thestarting point of translation (about 0.35 kb at the upstream of theinitiation point of transcription) was amplified by the combination ofPALRVXba and PALFSHin, and a promoter region (PAL-L) of about 1.5 kb bythe combination of PALRVXba and PALFFHin. The above-mentionedagrobacteruium plasmid DNA was used as a template and PCR was conductedwith these primers. The reaction conditions of PCR were as below:reaction solution: 50 μl; each primer: 0.5 μM, dNTP: 0.2 mM; 1×ExTAqbuffer, ExTAq DNA polymerase (from Takara Shuzo): 1U; and the reactionwas conducted once at 94° C. for three minutes, then 30 cycles at 94° C.for one minute, at 50° C. for one minute and at 72° C. for two minutes,and once at 72° C. for 6 minutes. A PCR product was cloned to vectorpCR11 (from Invitrogen).

[0047] Since the PsPAL1 promoter had a HinIII site at the upstream 142bp from the starting point of translation, PAL-S was digested completelywith restriction enzyme XbaI and then partially with HindIII to obtain a0.45 kb of fragment from pCR11. The above mentioned pSB21 was digestedwith HindIII and XbaI to remove the 35S promoter, and PAL-S wasintegrated thereinto. In the pSB21 vector employed here the unique PvuIIsite existing in the basic structure had been removed, and, instead, aPvuII linker had been placed at the unique ECOR1 site (just after theNos terminator). The plasmid with PAL-S integrated thereinto was furtherdigested with XbaI and SacI to remove the GUS gene, and then the abovementioned 1.1 kb hrpZ XbaI-SacI1 fragment was inserted therein.PALS-hrpZ was constructed according to the above procedure. Next, PAL-Lintegrated into pCR11 was digested with restriction enzymes XhoI andXbaI to take out a 1.45 kb PAL promoter, which was integrated intovector pSB11 (Komari et al.: Plant J. 10: 165-174 (1996)) co-digestedwith the same enzymes. The formed plasmid was digested with XbaI andSmaI, and an XbaI-PvuII fragment of PALS-hrpZ (hrpZ-NOS terminator) wasinserted therein. In this manner, PALL-hrpZ was produced. The PALpromoter promotes a low-level expression constitutively, but it is apromoter strongly induced with a pathogen and an injury (Yamada et al.:Plant Cell Physiol. 35: 917-926 (1994), and Kawamata et al.: Plant CellPhysiol. 38: 792-803 (1997)), and it is anticipated that a tobacco planttransformed with PALS-hrpZ or PALL-hrpZ accumulates more harpin_(pss) atthe place of stress when these stresses occur. In this case, it isanticipated that more harpin_(pss) will accumulate in the case of PALLrelative to the case of PALS.

[0048] According to the tri-parental mating system, of Escherichia coliLB392 strain containing the thus produced four constructs 35S-hrpZ,PALS-hrpZ, PALS-hrpZ and PALL-hrpZ (summarized in FIG. 1), agrobacteriumLBA4404 strain containing a vector pSB4U with a selection marker geneintegrated thereinto (corn ubiquitin promoter-hygromycin-resistant gene(hptII)-NOS terminator) and Escherichia coli HB101 containing a helperplasmid pRK2013, the hrpZ gene containing construct was introduced intoan agrobacterium utilizing homologous recombination.

[0049] The transformation of a tobacco was performed by the leaf discmethod (Horsch et al.: Science 227: 1229-1231 (1985)). A leaf of tobaccovariety SR1 grown in a greenhouse was sterilized by treatment withethanol for 30 seconds and with antiformin diluted 5 times for 5minutes, and after it was cleaned with sterilized water twice, it wascut into one-centimeter squares, and an agrobacterium suspension wasinoculated thereto. The concentrations of hygromycin at the time ofinduction and selection of a transfected shoot and at the time ofrooting were 50 or 100 mg/ml and 0 or 50 mg/ml, respectively. For thetransformation of rice, immature-embryo-derived cali of varieties ofpaddy rice, Tsukinohikari, and Koshihikari were transformed employingagrobacterium according to the method of Hiei et al.: Plant J. 6:271-282 (1994).

Example 4 Analysis of Transformants

[0050] (1) Transgenic Tobacco

[0051] 15 individuals of the re-generated plant were obtained from35S-hrpZ, 10 individuals were from PALS-hrpZ and 16 individuals werefrom PALL-hrpZ. There was observed no remarkable difference between theconstructs in transformation efficiency. Western analysis was performedon the primary generation (To) of the transformant, and Western analysisand disease assays were performed on the self-pollinated next generation(T₁).

[0052] 1) Western Analysis of T₀ Generation

[0053] 2×2 cm of a leaf of a transgenic tobacco of the 4 or 5 leaf stageand 2×2 cm of a leaf of a non-transgenic tobaco (SR1) were pulverized in0.1 M HEPES-KOH pH 7.6 buffer in a mortar. The supernatant liquid aftercentrifugation with 15000 g for 10 minutes was made a protein sample.The amount of the protein was determined with a Bio-Rad Protein Assaykit (from BIO-RAD). About 20 μg of the protein was fractioned by theSDS-PAGE method according to the method of Laemmni et al. (Nature 227:680-685 (1970)), on 12.5% PAGEL (from ATTO). After electrophoresis, theprotein bands on the gel were transferred to a PVDF membrane (fromMillipore). The PVDF membrane was placed in a 1×TBS buffer containing0.5% skim milk for 30 minutes, and shaken in the same buffer containing1/1000 (v/v) of anti-harpin_(pss) serum at room temperature overnight.As a secondary antibody was employed an anti-goat rabbit IgG peroxidaselabeled conjugate (from MBL) or an anti-goat rabbit IgG alkalinephosphatase conjugate (from BIO-RAD) at the concentration of 1/1000(v/v). As color development systems were employed HRP Color DevelopmentReagent (from BIO-RAD), alkaline phosphatase substrate kit II (fromVector Laboratories). The amounts of the protein expressed werecalculated by comparison with the color development of the harpin_(pss)sample of a known concentration, by using a densitometer (model GS-670,from BIO-RAD). Some of the results of the Western analysis of the T₀generation is shown in FIG. 2, and the whole results are summarized inTable 1.

[0054] The expression level is shown in four stages (+++, ++, +, −),which show 0.1% or more of the total soluble proteins (+++), 0.05 to0.1% (++), 0.05% or less (+) and below the detection limitation (−) inthe amount of expression, respectively. This is true also in Tables 2, 3and 4 to be described later. TABLE 1 Results of the Western Analysis ofthe Tobacco T₀ Generation Number of re-generated Expression level ofHarpin_(pss) ^(a) Construct individuals − + ++ +++^(b) PALS-hrpZ 10 1 81 0 PALL-hrpZ 16 2 10  4 0 35S-hrpZ 15 6 2 1 6 SR1 3 0 0 0

[0055] In the case of the constructs having a PAL promoter, theaccumulation of harpin_(pss) was detected in 80% or more of individuals.As anticipated, PALL had a larger proportion of high-expressionindividuals (++) than PALS. On the other hand, in the case of theconstruct having a 35S promoter, though no accumulation of harpin_(pss)was detected in 6 individuals of the 15 individuals, high-expressionindividuals were obtained in 7 individuals, near half of the totalindividuals. Besides, a very high expression (+++) was shown in 6individuals. Interestingly, no morphological change was observed in theorgan of any of a leaf, a stem, a root or a flower of thesehigh-expression individuals, and seed fertility was normal in almost allof them.

[0056] 2) Western Analysis of the T₁ Generation and Disease ResistanceAssay

[0057] Reaction to powdery mildew fungus (Erysiphe cichoracearum) wasanalized in about 8 lines of KH1-2 (PALS-hrpZ), KC6-7 (PALL-hrpZ), KC8-1(PALL-hrpZ), KK1-1 (35S-hrpZ), KK3-8 (35S-hrpZ), KK4-2 (35S-hrpZ), KK4-3(35ShrpZ), KK7-6 (35S-hrpZ), in which the amount of harpin_(pss)accumulated was high in the T₀ generation.

[0058] Tobacco individuals in which harpin_(pss) was accumulated at ahigh level in the T₀ generation were selected, and seeds ofself-pollinated next generation (T₁) thereof were obtained. The seedswere sowed and observed for about two months, but no visualmorphological change was observed for this period; they grew normally inthe same manner as the To generation, and no hypersensitive response wasobserved on the surface of a leaf. Then, powdery mildew fungi weresprayed to inoculate upon the T₁ generation of the transgenic tobacco ofthe 4 or 5 leaf stage and a disease resistance assay was performed.About 2 L of a suspension of powdery mildew fungi spores (1.4×106spores/ml) was spray-inoculated to 244 recombinants and 41 originalindividuals. As a result, hypersensitive-response-like localizednecrosis spots were induced onto a lower leaf of the recombinant 4 or 5days after inoculation (FIG. 3A, B). Surprisingly, not only in the caseof the PAL-hrpZ constructs but also in the case of the 35S-hrpZconstructs employing a constitutive promoter, specific localizednecrosis spots were induced after the pathogen infection (FIG. 3B). Theexpression frequency of localized necrosis spots on the 5th day afterthe inoculation was about 5% in the non-transformants, but the frequencywas from 6 to 14 times grater in the 35S-hrpZ construct (30 to 71%),from 4 to 5 times greater in the PAL-hrpZ constructs (20 to 27%) (Table2), and thereafter, in the case of the PAL-hrpZ constructs, the numberof local necrosis spots gradually increased. This was assumed to be dueto the response of the PsPAL1 promoter to Erysiphe cichoracearum. Thoughthe amount of harpin_(pss) accumulated and the degree of the formationof localized necrosis spots tended to be positively correlative (Table3), there were some exceptional transformants in which no accumulationof harpin_(pss) was detected at least in our Western analysis butlocalized necrosis spots occurred.

[0059] Next, in order to examine whether the localized necrosis spotshaving occurred after the powdery mildew infection were related todisease resistance, the symptom of powdery mildew on the 11th day afterthe inoculation thereof was examined. As a result, while there existedno individual in which the spread of powdery mildew hyphae was preventedin the non-transformants, from 15 to 57% individuals in the case of35S-hrpZ constructs and from 13 to 18% individuals in the case ofPAL-hrpZ constructs showed apparently less significant symptom ascompared to the non-transformants (FIG. 4, Table 2). The prevenstion ofthat the spread of powdery mildew was observed not only in leaves withlocalized necrosis spots but also in middle or upper leaves with nolocalized necrosis spots, and this is thought to be due to systemicacquired resistance (SAR). As a result of observing the hyphae ofpowdery mildew by cotton blue dyeing, the hyphae of powdery mildewextended sharply and spread around the surface in infested leaves of theSR1 of the original line as a control, whereas, though haustorium isformed on the surface of a leaf in the transformants, the spreading ofhyphae was prevented and stopped halfway. The promoters employed in thepresent studies are 35S promoter (constitutive) and PAL promoter(inducible); and it was found that when 35S promoter was employedinstead of PAL promoter, the frequency of localized necrosis spots washigher, and it was further found that at least according to examinationon the 11th day after inoculation, more individuals with a strongdisease resistance were obtained (Table 2). However, it was observedthat, in the case of employing the 35S promoter, the localized necrosisspots formed in response to the pathogen became larger (occupying 10% ormore of the leaf area) in some individuals, and as a result, lowerleaves died out. In addition, inversely, in some individuals withharpin_(pss) accumulated therein, localized necrosis spots were notobservable by the naked eye (Table 2), but some of such individuals hadresistance to powdery mildew (of individuals with − of localizednecrosis spots in Table 2, individuals of the number in parentheses; theamount of harpin_(pss) expressed is ++ in all). This is thought to beprobably due to the occurrence of a hypersensitive response in verysmall range, and it is possible that a disease-resistant plant with ahigh practicability can be obtained by the selection of suchindividuals. According to the fact that no localized necrosis spotoccurred without the invasion of the pathogen even in the case where thetransription of hrpZ gene was controlled with a constitutive promoter,it is possible to deduce that, since harpin_(pss) was recognized on theoutside of a transmembrane or cell wall of plant cells, probablyharpin_(pss) accumulated in cytoplasm was not recognized for plant cellstill the degradation of cells due to the invasion of the fungi, and as aresult, it caused a hypersensitive response after the inoculation of thepathogen. Another possibility may be that the elicitor activity ofharpin_(pss) requires the existence of some other factors derived fromthe pathogen or the plant, induced by the inoculation of the pathogen.TABLE 2 Relationship among the Amount of harpin_(pss) Accumulated, theFormation of Localized Necrosis Spots and Disease Resistance of theTobacco T₁ Generation Expression level Number of Line Name Construct(T₀) individuals analyzed (T₁) KH1-2 PALS-hrpZ ++ 18 KC6-7 PALL-hrpZ ++43 KC8-1 PALL-hrpZ ++ 44 KK1-1 35S-hrpZ +++ 23 KK3-8 35S-hrpZ +++ 33KK4-2 35S-hrpZ ++ 35 KK4-3 35S-hrpZ +++  7 KK7-6 35S-hrpZ +++ 41 SR1(control) − 41 Number of individuals with Rate of individuals Rate ofindividuals localized necrosis spots with localized with less progress(Number of individuals with necrosis spots of disease spots lessprogress of disease spots) (5th day after (11th day after Line Name +++++ + −^(a) inoculation) inoculation) KH1-2(PALS) 0 0  5(3) 13(0) 27% 16%KC6-7(PALL) 0 1(1)  8(6) 34(1) 20% 18% KC8-1(PALL) 0 1(0) 11(5) 32(1)27% 13% KK1-1(35S) 0 0  7(3) 16(1) 30% 17% KK3-8(35S) 0 2(0) 11(5) 20(0)39% 15% KK4-2(35S) 1(1) 4(3) 15(6) 15(0) 57% 28% KK4-3(35S) 0 3(3)  2(1) 2(0) 71% 57% KK7-6(35S) 1(1) 4(4) 18(4) 18(1) 56% 24% SR1 (control) 0 0 2(0) 39(0)  5%  0%

[0060] TABLE 3 Relationship between the Expression level of Harpin_(pss)and the Number of Localized Necrosis Spots in the Tobacco T₁ GenerationIncidence of Expression level localized of harpin_(pss) ^(a) Degree oflocalized necrosis spots^(b) necrosis (Western analysis) +++ ++ + −spots +++ 1 4 19 19 56% ++ 0 5 32 77 32% + 1 6 18 38 40% − 0 1  5 18 25%SR1 0 0  2 39  5%

[0061] (2) Transgenic Rice

[0062] 1) Western Analysis of the T₀ Generation

[0063] Harpin_(pss) was introduced into a rice variety, Tsukinohikari.35 individuals of the regenerated plant were obtained from the 35S-hrpZconstruct, and 26 individuals of the regenerated plant were obtainedfrom the PPDK-hrpZ construct. There was observed no remarkabledifference between the constructs in transformation efficiency. Westernanalysis was performed on the primary generation (T₀) of thetransformation and individuals with a high expression were selected.

[0064] Protein was extracted from the regenerated transgenic rice(Tsukinohikari) in the same manner as in the example of the tobacco andsubjected to Western analysis. The results of Western analysis of the T₀generation are shown in Table 4. TABLE 4 Results of the Western Analysisof the T₀ Generation of Rice (Tsukinohikari) Number of regeneratedExpression level of harpin_(pss) ^(a) Construct individuals − + +++++^(b) 35S-hrpZ 35 17  5 13 0 PPDK-hrpZ 26  9 13  4 0

[0065] In the case of the rice (Tsukinohikari), similar to the case ofthe tobacco, individuals with a high-expression of harpin_(pss) wereobtained (see also FIG. 2). In the case of a construct having a 35Spromoter, the accumulation of harpin_(pss) was detected in about half ofthe individuals, and the rate of high-expression individuals (++) wasabout one-third or more of the whole. Also, in the case of a PPDKpromoter the accumulation of harpin_(pss) was detected in abouttwo-thirds of the individuals, and of them, 4 individuals showed a highexpression. Interestingly, no morphological change was observed in theorgan of any of a leaf, a root or a flower of these high-expressionindividuals. And seed fertility was normal in almost all of them, and T₁seeds of high-expression individuals could be obtained.

[0066] 2) Western Analysis of the T₀ Generation and the DiseaseResistance Assay of the T₁ Generation

[0067] Next, harpin_(pss) was introduced into Koshihikari, one of themost important varieties of rice of Japan. The results of the Westernanalysis of the T₀ generation are shown in Table 5. TABLE 5 Results ofthe Western Analysis of the T₀ Generation of Rice (Koshihikari) Numberof regenerated Expression level of harpin_(pss) ^(a) Constructindividuals − + ++ +++^(b) 35S-hrpZ 78 18 33 21 6 PPDK-hrpZ 27  7 13  70

[0068] Of the individuals of the T₀ generation with the 35ShrpZconstruct introduced thereinto, four individuals showning a large amount(+++ in Table 5) of the accumulation of harpin_(pss) (hrp5-8, hrp23-5,hrp24-1, hrp429) were selected, and their vulnerability to rice blast inthe T₁ generation was examined. The seed fertility of the selected fourhigh-expression individuals was normal, and many self-fertilized seedscould be obtained. T₁ seeds were sowed in a seedling case with culturesoil in a manner of 8 seeds×2 rows, cultivated in a greenhouse, andsubjected to a disease assay at the 4.8 to 5.2 leaf stage. As a riceblast fungus (Magneporthe grisea) was employed race 007. Forinoculation, a conidium formed by culturing the blast fungi on anoatmeal sucrose agar medium at 28° C. under dark condition and then,after the spread of the fungi, at 25° C., irradiating near ultravioletlight for three days was employed. The inoculation of the blast fungiwas performed by spray-inoculating 30 ml of a suspension adjusted to1.5×10⁵ condia/ml in 0.02% Tween 20 per three seedling cases. Thespray-inoculated rice was held in a moistening incubator (SLPH-550-RDS,manufactured by Nippon Medical & Chemical Instruments Co. Ltd.) for 24hours after the inoculation at 25° C. at a humidity of 100%, and thentransferred into the greenhouse. The conditions of the greenhouse wereset at 25° C. under light conditions for 16 hours, and at 22° C. underdark conditions for 8 hours. The evaluation of disease resistance wasperformed by visually counting the number of progressive disease spotson the 5th leaf at 6th day after the inoculation, said leaf being thetopmost development leaf at the time of inoculation. Significantdifferences among the results were evaluated according to theMann-Whitney U test.

[0069] As a result, though no localized necrosis spot due to theinoculation of the blast fungi was observed, the average number ofprogressive disease spots was reduced by 24 to 38% relative to thecontrol Koshihikari in three lines (hrp5-8, hrp42-9, hrp23-5) out of thefour lines of the harpin_(pss)-introduced rice. Moreover, this reductionwas statistically significant (Table 6). The above results show that thedisease resistance of rice could be increased by the introduction ofharpin_(pss). TABLE 6 Results of the Disease Test against Rice Blast ofthe Four Lines of Harpin_(pss)- Intorduced Rice (T₁ Generation) Numberof Number of average tested progressive disease spots^(a) Strainindividuals (standard error) Significant Test^(b) hrp5-8 16  9.3 (±1.0)significant (significance level 1%) hrp23-5 21 11.4 (±1.3) significant(significance level 5%) hrp24-1 20 14.4 (±1.4) No significant differencehrp42-9 14  9.4 (±1.4) significant (significance level 1%) Koshihikari64 15.0 (±0.7) —

[0070] As a result of the present invention, it has become apparent forthe first time that disease resistance can be imparted to a plant byconnecting a gene enconding harpin to a constitutive promoter or aninducible promoter and introducing the gene into the plant. Thisharpinin-introduced plant is thought to be useful for explicating thefunction of harpin as a protein elicitor, and also for explicating themechanism of localized or systemic acquired resistance. In addition, itis revealed that the production of a harpin-introduced resistant plant,which has been thought to be difficult without the use of an induciblepromoter, can sufficiently be realized by employing a constitutivepromoter, and the extension of the application range of the presentapproach can be shown. The present invention shows that a method forproducing a disease-resistant plant by integrating a DNA sequenceencoding a harpin into an expression cassette comprising a sequence ofan appropriate constitutive, or organ- or phase-specific promotercapable of functioning in a plant cell, or a promoter induced withstress or pests, and a sequence of a terminator capable of functioningin a plant cell, and introducing it into the plant cell to obtain aregenerated individual is a useful and effective approach in view ofgenetic engineering.

1 7 1 1029 DNA Pseudomonas syringae pv. syringae LOB2-1 1 atg cag agtctc agt ctt aac agc agc tcg ctg caa acc ccg gca atg 48 Met Gln Ser LeuSer Leu Asn Ser Ser Ser Leu Gln Thr Pro Ala Met 1 5 10 15 gcc ctt gtcctg gta cgt cct gaa acc gag acg act ggc gcc agt acg 96 Ala Leu Val LeuVal Arg Pro Glu Thr Glu Thr Thr Gly Ala Ser Thr 20 25 30 tcg agc aag gcgctt cag gaa gtt gtc gtg aag ctg gcc gag gaa ctg 144 Ser Ser Lys Ala LeuGln Glu Val Val Val Lys Leu Ala Glu Glu Leu 35 40 45 atg cgc aat ggt caactc gac gac agc tcg cca ttg ggc aaa ctg ctg 192 Met Arg Asn Gly Gln LeuAsp Asp Ser Ser Pro Leu Gly Lys Leu Leu 50 55 60 gcc aag tcg atg gcc gcggat ggc aag gca ggc ggc ggt atc gag gat 240 Ala Lys Ser Met Ala Ala AspGly Lys Ala Gly Gly Gly Ile Glu Asp 65 70 75 80 gtc atc gct gcg ctg gacaag ctg att cat gaa aag ctg ggt gac aac 288 Val Ile Ala Ala Leu Asp LysLeu Ile His Glu Lys Leu Gly Asp Asn 85 90 95 ttc ggc gcg tct gcg gac aacgcc tcg ggt acc gga cag cag gac ctg 336 Phe Gly Ala Ser Ala Asp Asn AlaSer Gly Thr Gly Gln Gln Asp Leu 100 105 110 atg act cag gtg ctc agt ggcctg gcc aag tct atg ctc gat gat ctt 384 Met Thr Gln Val Leu Ser Gly LeuAla Lys Ser Met Leu Asp Asp Leu 115 120 125 ctg acc aag cag gat ggc ggggca agc ttc tcc gaa gac gat atg ccg 432 Leu Thr Lys Gln Asp Gly Gly AlaSer Phe Ser Glu Asp Asp Met Pro 130 135 140 atg ctg aac aag atc gcg cagttc atg gat gac aat ccc gca cag ttt 480 Met Leu Asn Lys Ile Ala Gln PheMet Asp Asp Asn Pro Ala Gln Phe 145 150 155 160 ccc aag ccg gac tcg ggttcc tgg gtg aac gaa ctc aag gaa gac aac 528 Pro Lys Pro Asp Ser Gly SerTrp Val Asn Glu Leu Lys Glu Asp Asn 165 170 175 ttc ctt gat ggc gac gaaacg gct gcg ttc cgc tcg gca ctc gac atc 576 Phe Leu Asp Gly Asp Glu ThrAla Ala Phe Arg Ser Ala Leu Asp Ile 180 185 190 att ggc cag caa ctg ggtaat cag cag agt ggc gct ggc ggt ctg gcg 624 Ile Gly Gln Gln Leu Gly AsnGln Gln Ser Gly Ala Gly Gly Leu Ala 195 200 205 ggg acg ggt gga ggt ctgggc act ccg agc agt ttt tct aac aac tcg 672 Gly Thr Gly Gly Gly Leu GlyThr Pro Ser Ser Phe Ser Asn Asn Ser 210 215 220 tcc gtg acg ggt gat ccgctg atc gac gcc aat acc ggt ccc ggt gac 720 Ser Val Thr Gly Asp Pro LeuIle Asp Ala Asn Thr Gly Pro Gly Asp 225 230 235 240 agc ggc aat agc agtggt gag gcg ggg caa ctg atc ggc gag ctt atc 768 Ser Gly Asn Ser Ser GlyGlu Ala Gly Gln Leu Ile Gly Glu Leu Ile 245 250 255 gac cgt ggc ctg caatcg gta ttg gcc ggt ggt gga ctg ggc aca ccc 816 Asp Arg Gly Leu Gln SerVal Leu Ala Gly Gly Gly Leu Gly Thr Pro 260 265 270 gta aac acc ccg cagacc ggt acg gcg gcg aat ggc gga cag tcc gct 864 Val Asn Thr Pro Gln ThrGly Thr Ala Ala Asn Gly Gly Gln Ser Ala 275 280 285 cag gat ctt gac cagttg ctg ggc ggc ttg ctg ctc aag ggc ctt gaa 912 Gln Asp Leu Asp Gln LeuLeu Gly Gly Leu Leu Leu Lys Gly Leu Glu 290 295 300 gcg acg ctc aag gatgcc ggt caa acc gct acc gac gtg cag tcg agc 960 Ala Thr Leu Lys Asp AlaGly Gln Thr Ala Thr Asp Val Gln Ser Ser 305 310 315 320 gct gcg caa atcgcc acc ttg ctg gtc agt acg ctg ctg caa ggc acc 1008 Ala Ala Gln Ile AlaThr Leu Leu Val Ser Thr Leu Leu Gln Gly Thr 325 330 335 cgc aat cag gctgca gcc tga 1029 Arg Asn Gln Ala Ala Ala 340 2 342 prt Pseudomonassyringae pv. syringae LOB2-1 2 Met Gln Ser Leu Ser Leu Asn Ser Ser SerLeu Gln Thr Pro Ala Met 1 5 10 15 Ala Leu Val Leu Val Arg Pro Glu ThrGlu Thr Thr Gly Ala Ser Thr 20 25 30 Ser Ser Lys Ala Leu Gln Glu Val ValVal Lys Leu Ala Glu Glu Leu 35 40 45 Met Arg Asn Gly Gln Leu Asp Asp SerSer Pro Leu Gly Lys Leu Leu 50 55 60 Ala Lys Ser Met Ala Ala Asp Gly LysAla Gly Gly Gly Ile Glu Asp 65 70 75 80 Val Ile Ala Ala Leu Asp Lys LeuIle His Glu Lys Leu Gly Asp Asn 85 90 95 Phe Gly Ala Ser Ala Asp Asn AlaSer Gly Thr Gly Gln Gln Asp Leu 100 105 110 Met Thr Gln Val Leu Ser GlyLeu Ala Lys Ser Met Leu Asp Asp Leu 115 120 125 Leu Thr Lys Gln Asp GlyGly Ala Ser Phe Ser Glu Asp Asp Met Pro 130 135 140 Met Leu Asn Lys IleAla Gln Phe Met Asp Asp Asn Pro Ala Gln Phe 145 150 155 160 Pro Lys ProAsp Ser Gly Ser Trp Val Asn Glu Leu Lys Glu Asp Asn 165 170 175 Phe LeuAsp Gly Asp Glu Thr Ala Ala Phe Arg Ser Ala Leu Asp Ile 180 185 190 IleGly Gln Gln Leu Gly Asn Gln Gln Ser Gly Ala Gly Gly Leu Ala 195 200 205Gly Thr Gly Gly Gly Leu Gly Thr Pro Ser Ser Phe Ser Asn Asn Ser 210 215220 Ser Val Thr Gly Asp Pro Leu Ile Asp Ala Asn Thr Gly Pro Gly Asp 225230 235 240 Ser Gly Asn Ser Ser Gly Glu Ala Gly Gln Leu Ile Gly Glu LeuIle 245 250 255 Asp Arg Gly Leu Gln Ser Val Leu Ala Gly Gly Gly Leu GlyThr Pro 260 265 270 Val Asn Thr Pro Gln Thr Gly Thr Ala Ala Asn Gly GlyGln Ser Ala 275 280 285 Gln Asp Leu Asp Gln Leu Leu Gly Gly Leu Leu LeuLys Gly Leu Glu 290 295 300 Ala Thr Leu Lys Asp Ala Gly Gln Thr Ala ThrAsp Val Gln Ser Ser 305 310 315 320 Ala Ala Gln Ile Ala Thr Leu Leu ValSer Thr Leu Leu Gln Gly Thr 325 330 335 Arg Asn Gln Ala Ala Ala 340 3 30DNA Artificial Sequence Synthetic primer derived from Pseudomonassyringae pv. Syringae 61 3 aaaatctaga atgcagagtc tcagtcttaa 30 4 30 DNAArtificial Sequence Synthetic primer derived from Pseudomonas syringaepv. Syringae 61 4 aaaagtcgac tcaggctgca gcctgattgc 30 5 32 DNAArtificial Sequence Synthetic primer derived from the reported PSPAL1promoter 5 ggggtctaga attgatacta aagtaactaa tg 32 6 32 DNA ArtificialSequence Synthetic primer derived from the reported PSPAL1 promoter 6ttggaagctt agagatcatt acgaaattaa gg 32 7 29 DNA Artificial SequenceSynthetic primer derived from the reported PSPAL1 promoter 7 ctaaaagcttggtcatgcat ggttgcttc 29

1. A transgenic, disease-resistant plant which has been transformed withan expression cassette comprising: a promoter capable of promoting aconstitutive, inducible, or organ- or phase-specific gene expression;and a gene, under the control of said promoter, encoding an elicitorprotein; wherein said plant is capable of effecting the constitutive,inducible, or organ- or phase-specific expression of the elicitorprotein in an amount effective for inducing a defense reaction.
 2. Atransgenic, disease-resistant plant as claimed in claim 1, wherein saidpromoter capable of promoting a constitutive, inducible, or organ- orphase-specific gene expression and said gene, under the control of saidpromoter, encoding an elicitor protein, are integrated into the genome.3. A transgenic, disease-resistant plant as claimed in claim 1 or 2,wherein said elicitor protein is a protein possessing ahypersensitive-response-inducing activity against diseasemicroorganisms.
 4. A transgenic, disease-resistant plant as claimed inclaim 3, wherein said protein possessing ahypersensitive-response-inducing activity is selected from: (a) aprotein consisting of the amino acid sequence of SEQ. ID No. 2; (b) aprotein consisting of an amino acid sequence derived from the amino acidsequence of SEQ. ID No. 2 by deletion, substitution, addition orinsertion of one or more amino acids, and possessing ahypersensitive-response-inducing activity; and (c) a protein consistingof an amino acid sequence being at least 50% homologous to the aminoacid sequence of SEQ. ID No. 2, and possessing ahypersensitive-response-inducing activity.
 5. A transgenic,disease-resistant plant as claimed in claim 2, wherein said geneencoding an elicitor protein is selected from: (a) a DNA moleculeconsisting of the nucleotide sequence of SEQ. ID No. 1; (b) a DNAmolecule consisting of a nucleotide sequence derived from the nucleotidesequence of SEQ. ID No. 1 by deletion, substitution, addition orinsertion of one or more nucleotides, and encoding a protein possessinga hypersensitive-response-inducing activity; (c) a DNA moleculeconsisting of a nucleotide sequence being hybridizable with a DNAmolecule consisting of the complementary nucleotide sequence to thenucleotide sequence of SEQ. ID No. 1 under stringent conditions, andencoding a protein possessing a hypersensitive-response-inducingactivity; and (d) a DNA molecule consisting of a nucleotide sequencebeing at least 50% homologous to the nucleotide sequence of SEQ. ID No.1, and encoding a protein possessing a hypersensitive-response-inducingactivity.
 6. A method for producing a transgenic, disease-resistantplant capable of effecting a constitutive, inducible, or organ- orphase-specific expression of an elicitor protein in an amount effectivefor inducing a defense reaction, comprising the steps of: (a) obtaininga transgenic plant cell with an expression cassette comprising apromoter capable of promoting a constitutive, inducible, or organ- orphase-specific gene expression and a gene, under the control of saidpromoter, encoding an elicitor protein; and (b) reconstructing, fromsaid transgenic plant cell, a complete plant.
 7. An expression cassettefor producing a transgenic, disease-resistant plant capable of effectinga constitutive, inducible, or organ- or phase-specific expression of anelicitor protein in an amount effective for inducing a defense reaction,comprising at least: (a) a promoter capable of promoting a constitutive,inducible, or organ- or phase-specific gene expression; and (b) a gene,under the control of said promoter, encoding the elicitor protein.
 8. Anexpression cassette as claimed in claim 7, wherein said elicitor proteinis a protein possessing a hypersensitive-response-inducing activityagainst disease microorganisms.
 9. An expression cassette as claimed inclaim 8, wherein said protein possessing ahypersensitive-response-inducing activity is selected from: (a) aprotein consisting of the amino acid sequence of SEQ. ID No. 2; (b) aprotein consisting of an amino acid sequence derived from the amino acidsequence of SEQ. ID No. 2 by deletion, substitution, addition orinsertion of one or more amino acids, and possessing ahypersensitive-response-inducing activity; and (c) a protein consistingof an amino acid sequence being at least 50% homologous to the aminoacid sequence of SEQ. ID No. 2, and possessing ahypersensitive-response-inducing activity.
 10. An expression cassette asclaimed in claim 7, wherein said gene encoding an elicitor protein isselected from: (a) a DNA molecule consisting of the nucleotide sequenceof SEQ. ID No. 1; (b) a DNA molecule consisting of a nucleotide sequencederived from the nucleotide sequence of SEQ. ID No. 1 by deletion,substitution, addition or insertion of one or more nucleotides, andencoding a protein possessing a hypersensitive-response-inducingactivity; (c) a DNA molecule consisting of a nucleotide sequence beinghybridizable with a DNA molecule consisting of the complementarynucleotide sequence to the nucleotide sequence of SEQ. ID No. 1 understringent conditions, and encoding a protein possessing ahypersensitive-response-inducing activity; and (d) a DNA moleculeconsisting of a nucleotide sequence being at least 50% homologous to thenucleotide sequence of SEQ. ID No. 1, and encoding a protein possessinga hypersensitive-response-inducing activity.
 11. An expression cassetteas claimed in any one of claims 7-10 for producing a transgenic,systemic acquired disease-resistant plant.
 12. An expression cassette asclaimed in any one of claims 7-11, wherein said elicitor protein isexpressed specifically at the time of infection of diseasemicroorganisms in an amount effective for inducing a defense reaction.13. An expression cassette as claimed in claim 12, comprising aconstitutive, or organ- or phase-specific promoter.
 14. A recombinantvector carrying an expression cassette as claimed in any one of claims7-13.
 15. A gene consisting of a DNA molecule selected from: (a) a DNAmolecule consisting of the nucleotide sequence of SEQ. ID No. 1; (b) aDNA molecule consisting of a nucleotide sequence derived from thenucleotide sequence of SEQ. ID No. 1 by deletion, substitution, additionor insertion of one or more nucleotides, and encoding a proteinpossessing a hypersensitive-response-inducing activity; (c) a DNAmolecule consisting of a nucleotide sequence being hybridizable with aDNA molecule consisting of the complementary nucleotide sequence to thenucleotide sequence of SEQ. ID No. 1 under stringent conditions, andencoding a protein possessing a hypersensitive-response-inducingactivity; and (d) a DNA molecule consisting of a nucleotide sequencebeing at least 50% homologous to the nucleotide sequence of SEQ. ID No.1, and encoding a protein possessing a hypersensitive-response-inducingactivity.
 16. A gene encoding a protein selected from: (a) a proteinconsisting of the amino acid sequence of SEQ. ID No. 2; (b) a proteinconsisting of an amino acid sequence derived from the amino acidsequence of SEQ. ID No. 2 by deletion, substitution, addition orinsertion of one or more amino acids, and possessing ahypersensitive-response-inducing activity; and (c) a protein consistingof an amino acid sequence being at least 97% homologous to the aminoacid sequence of SEQ. ID No. 2, and possessing ahypersensitive-response-inducing activity.
 17. A protein selected from:(a) a protein consisting of the amino acid sequence of SEQ. ID No. 2;(b) a protein consisting of an amino acid sequence derived from theamino acid sequence of SEQ. ID No. 2 by deletion, substitution, additionor insertion of one or more amino acids, and possessing ahypersensitive-response-inducing activity; and (c) a protein consistingof an amino acid sequence being at least 97% homologous to the aminoacid sequence of SEQ. ID No. 2, and possessing ahypersensitive-response-inducing activity.
 18. A transgenic,disease-resistant plant as claimed in any one of claims 1-5, which hasbeen transformed with an expression cassette comprising a constitutiveor inducible promoter; wherein said plant is a transgenic, powderymildew-resistant tobacco.
 19. A transgenic, disease-resistant plant asclaimed in any one of claims 1-5, which has been transformed with anexpression cassette comprising a constitutive promoter; wherein saidplant is a transgenic, blast-resistant rice.